THE EFFECT OF ARSENATE ON AEROBIC PHOSPHORYLATION

Transcription

1 THE EFFECT OF ARSENATE ON AEROBIC PHOSPHORYLATION BY ROBERT K. CRANE* AND FRITZ LIPMANN (From the Biochemical Research Laboratory, Massachusetts General Hospital, and the Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts) (Received for publication, October 1, 1952) Effects of arsenate on the phosphate turnover in cell-free alcoholic fermentation were first observed by Harden (1). Subsequently, Needham and Pillai (2) found that the phosphate requirement in the phosphoglyceraldehyde dehydrogenation system can be satisfied by arsenate. More recently, Warburg and Christian (3) proposed that the observed effects of arsenate were due to an instantaneous hydrolysis of arsenate esters in contrast to the relative stability of their phosphate counterparts. They arrived at this conclusion after they had found that phosphoglyceraldehyde in the presence of phosphate was oxidized to phosphoglyceryl phosphate, while arsenate produced free phosphoglycerate as the oxidation product. It was implied from this observation that phosphoglyceryl arsenate spontaneously decomposes. Doudoroff, Barker, and Hassid (4) extended this concept when they found that arsenate together with sucrose phosphorylase caused the hydrolysis of glucose-l-phosphate. They introduced the term arsenolysis for this phenomenon. An analogous arsenolysis of acetyl phosphate with transacetylase has been reported by Stadtman and Barker (5). The mechanism of this reaction was later studied by Stadtman in this laboratory (6). In view of the rather general property of arsenate to disrupt phosphorylation reactions by substitution for phosphate and eventual formation of unstable arsenyl compounds, it appeared of considerable interest to explore the effect of arsenate on aerobic phosphate bond generation in mitochondrial suspensions. Methods Preparation of Enzyme-A procedure similar to that of Green, Loomis, and Auerbach (7) and Loomis and Lipmann (8) was used routinely for the preparation of a particulate enzyme fraction from rat liver. Adult male albino rats were killed by stunning. The livers were excised and chilled in cracked ice. All further operations in the preparation of the enzyme were carried out in the cold, with previously chilled apparatus and reagents. The intact liver (6 to 10 gm. wet weight) was forced through a * Present address, Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri.. 235

2 236 AEROBIC PHOSPHORYLATION perforated stainless steel plate. The extruded pulp, essentially free of connective tissue, was collected and suspended in 100 ml. of a solution containing 0.9 per cent KC1 and 0.02 M NaHC03. The suspension was homogenized in a 500 ml. container with a I.5 ampere Waring blendor for 20 seconds. The insoluble fraction of this homogenate was separated by centrifugation at 2000 X g for 10 minutes. The supernatant fluid was discarded and the residue was washed twice with the KCl-NaHC08 by resuspension and sedimentation. The washed residue was suspended for manometric use in 30 ml. of the KCl-NaHC03 solution. This preparation contains, in addition to the predominant mitochondria, a very few unbroken cells, a large number of intact nuclei, and red cells. Experimental Conditions-All incubations were made at 30 in an atmosphere of air. The ph was 7.2. Other conditions were as noted in the legends to Figs. 1 and 2 and Tables I to V. Assay of P32; Separation of Inorganic Phosphate and Adenosinetriphosphate (ATP)-The use of P32 as a tool in detecting less obvious relationships between inorganic and ATP phosphorus has been hampered by the lack of a simple method for the quantitative separation of these entities. Separation has been previously accomplished by such procedures as precipitation of the inorganic phosphate in the form of the calcium salt (9) or the magnesium ammonium complex (10) by counter-current extraction (11) and chromatography on Dowex 1 (12). Neither of the precipitation methods is quantitative in the sense required for work with tracers. The solvent distribution procedure, though elegantly quantitative, is tedious, and resin chromatography is likewise rather time-consuming. We have adapted the property of certain charcoals to adsorb the adenosine-containing phosphate compounds without adsorbing inorganic phosphate (13) to this separation and have been able to achieve quantitative results with comparative ease. Though numerous modifications are immediately obvious, we have found the following procedure to be satisfactory. The experiment is terminated by the addition of trichloroacet,ic acid (TCA) (2 ml. of 10 per cent TCA to 3 ml. of enzyme incubation mixture). The acidified medium is made up to 10 ml. with 5 per cent TCA and centrifuged at 3000 X g for 10 minutes. The clear supernatant fluid is decanted from the insoluble residue. To 5 ml. of the supernatant fluid is added 0.2 to 0.3 gm. of Norit A charcoal, which is uniformly suspended in the solution by shaking or stirring. To reduce the amount of charcoal which floats, 0.2 ml. of 95 per cent ethanol is layered on the solution. Then the charcoal is sedimented by centrifugation at 1000 X g for 5 minutes. The supernatant fluid is decanted carefully and the charcoal residue is 1 This method was suggested to us by some unpublished observations of Dr. John 0. Hutchens. *

3 R. K. CRANE AND F. LIPMANN 237 washed twice by suspending in 5 ml. of water and centrifuging as before. Ethanol must be layered on the solution each time. The supernatant fluid and washings from the charcoal are combined as the inorganic phosphate fraction. After the second washing the tube is drained by inverting on absorbent paper. To recover the labile phosphorus of the nucleotide fraction, the charcoal residue is then suspended in 2 ml. of 1 N HCI and placed in a boiling water bath for 10 minutes.2 After cooling, the contents are made to a convenient volume with water, mixed thoroughly by inversion, and filtered to remove the charcoal. For assay of the radioactivity, small aliquots may be dried in Pyrex planchets or larger aliquots may be assayed in solution by dip counting (14). Analysis of the phosphate content as described below may be made directly on these solutions. In this case, it is best to neutralize the tubes containing HCl by the addition of 2 ml. of silicate-free 1 N NaOH. 14 study of the adsorptive properties of Norit A in regard to the more common biological compounds containing phosphate showed that the adenosine-containing phosphate compounds were the only ones of those tested which were adsorbed. Those compounds tested which were not at all adsorbed were inorganic orthophosphate, inorganic pyrophosphate, fructose diphosphate, glucose-l-phosphate, fructose-6-phosphate, and acetyl phosphate.3 It is presumed that other nucleotidic compounds are likewise adsorbed (13), although no specific tests were performed. These compounds, however, did not interfere appreciably in studies, like the present one, concerned mainly with the exchange of inorganic P32 and ATP. ATP and AMP (adenosine-5-phosphate) were quantitatively adsorbed and the labile phosphate of ATP was released quantitatively as inorganic phosphate by the treatment described above. Some release (approximately 50 per cent) of the non-labile phosphate of AMP and ATP from the charcoal does occur during the treatment with acid. However, this phosphate remains in an organic form and does not interfere with the analyses. Analysis of Phosphorus-Inorganic phosphorus and easily hydrolyzable phosphorus were estimated by the method of Fiske and Subbarow (15) as modified by Lohmann and Jendrassik (16). Since arsenate causes color formation in this method, all solutions containing arsenite or arsenate were treated by the method of Pett (17) before analysis for phosphorus. Since arsenate is not adsorbed on charcoal in the method of separation described above, the treatment for arsenate was applied to only the inorganic fraction and the easily hydrolyzable phosphate was determined by analysis of the filtrate from the charcoal hydrolysate. 2 The nucleotide fraction may be r&covered without hydrolysis by eluting with 5 per cent aqueous pyridine. 3 The observation on acetyl phosphate was made by Dr. H. Chantrenne.

4 238 AEROBIC PHOSPHORYLATION Results Substitution of Phosphate by Arsenate-It is well known through the work of a great variety of investigators (18) that the rate of oxygen uptake of washed mitochondria is dependent on the presence of inorganic phosphate. It may be seen from the experiment represented in Fig. 1 that arsenate is able to substitute for phosphate, causing a considerable increase in respiration of washed suspensions. It was noted, furthermore, that the addition of small amounts of phosphate to the arsenate-supplemented 120 too- MINUTES FIG. 1. The effect of arsenate on oxygen consumption at low concentrations of adenosine-5-phosphate and phosphate. The Warburg vessels contained, as final concentrations, 0.01 M L-glutamate, M MgC12, M tris(hydroxymethyl)- aminomethane buffer, M adenosine-5-phosphate, 1.0 ml. of the enzyme preparation, and, as indicated below, 0.01 M arsenate and M phosphate. 0.9 per cent KC1 was added to make a total of 2.8 ml. Curve A, either no additions or addition of phosphate only gave indistinguishable results; Curve B, arsenate only added; Curve C, both arsenate and phosphate added. respiration system caused a decrease in respiration. This interesting phenomenon has been very frequently observed, although no satisfactory explanation can be offered at the present. time. In the experiment, shown in Table I, it is seen that arsenate decreases the requirement for adenylic acid. This effect most likely should be due to an uncoupling of oxidation and phosphorylation. Before further studies are discussed, a complicating inhibition of respiration in the presence of arsenate needs consideration. This inhibition was rather variable, but frequently quite pronounced, and it, is almost certainly due to the appearance by reduction of arsenite and not to arsenate itself. Fig. 2 shows that the inhibition of oxygen consumption by arsenate,

5 R. K. CRANE AND F. LIPMANN 239 particularly at lower concentrations, is negligible at first, but increases markedly with time, the rate of increase being proportional to the arsenate concentration. Table II presents further data both on respiration and phosphorylation in the presence of arsenate. Glutamate, it will be noted, was used as TABLE Effect of Arsenate on Requirement for Phosphate and Adenosine-5-Phosphate to Support Oxygen Uptake I Additions NOIE Phosphate AMP ~.~ AMP phosphate Arsenate AMP arsenate O2 uptake, ~ The Warburg vessels contained, as final concentrations, 0.01 M L-glutamate, M MgC12, M tris(hydroxymethyl)aminomethane buffer, 1.0 ml. of the enzyme preparation, and, as indicated, 0.01 M arsenate, 0.01 M phosphate, and M adenosine-5-phosphate. 0.9 per cent KC1 was added to make a total of 2.8 ml. The additions were carried in the side arm during the temperature equilibration. Duration of measurement, 30 minutes. 0 5 IO M%UT:o FIG. 2. The inhibition of oxygen consumption by various concentrations of arsenate. The Warburg vessels contained, as final concentrations, 0.01 M L-glutamate, M MgC12, M tris(hydroxymethyl)aminomethane buffer, 0.02 M phosphate, M adenosinetriphosphate, 1.0 ml. of the enzyme preparation, and arsenate as shown below. 0.9 per cent KC1 was added to make a total of 2.8 ml. Arsenate was carried in the side arm during temperature equilibration. The points on the curves represent the per cent inhibition observed for the 5 minute period immediately preceding. Curve A, M arsenate; Curve B, nr; Curve C, M; Curve D, M; Curve E, M.

6 240 SEROBIC PHOSPHORYLATION substrate. It is seen that phosphorylation as well as oxygen consumption is progressively inhibited, but phosphorylation more markedly. In confirmation of our suspicion that this inhibition is due to arsenite, it was found that this system is extremely sensitive to arsenite and markedly inhibited with concentrations as low as 3 X 10-j M. TABLE Effect of Arsenate on Respiration and Phosphorylation As Function of Time II Time interval - I Oxygen uptake Control Arsenate I Per cent inhibition Control Phosphate Arsenate uptake Per cent inhibition patoms HI PM 12.3 / / The Warburg vessels contained, as final concentrations, 0.01 M n-glutamate, M MgC12, M tris(hydroxymethyl)aminomet~hane buffer, 0.02 M phosphate, r~ NaF, M adenosine-5-phosphate, 0.15 ml. of a yeast hexokinase preparation, M glucose, 1.0 ml. of the enzyme preparation, and, as indicated, M arsenate. 0.9 per cent KC1 was added to make a final volume of 2.8 ml. Arsenate, hexokinase, and glucose were carried in a side arm during temperature equilibration. TABLE III Effect of Arsenate and Arsenite on Oxygen Uptake and Phosphorylation with Succinate As Substrate O2 uptake, patoms.... P p&f... P:O ratsi... Arsenate The Warburg vessel contents were as for Table II, except that 0.01 M succinate replaced L-glutamate and, as indicated above, M arsenate and M arsenite were added. Duration, 15 minutes. In order to obviate, at least in part, the arsenite effect, which is presumably due primarily to inhibition of keto acid oxidation, experiment s with succinate as substrate were carried out. The results of such an experiment are shown in Table III. It may be seen that arsenite in this case causes much less inhibition. It is noteworthy that with the succinate as substrate the coupling with phosphorylation of the remaining oxygen uptake appears not to be affected by arsenite. This may be seen from the nearly equal P:O ratios with and without addition of inhibitor. In contrast, arsenate has very little if any effect on oxygen consumption; it does, how$ver, appreciably inhibit phosphorylation.

7 R. K. CRANE AND F. LIPMANN 241 Effect of Arsenate on Exchange of ATP with P32-For various reasons it was considered more advantageous to use-glutamate as the substrate and to incubate for very short periods of time. In this way conversion to arsenite could be held to low enough levels so that it would not interfere with the observations to be made. Various concentrations of arsenate were added to a system actively exchanging phosphate. The results, presented in Table IV, show marked reduction in specific activity of ATP phosphorus as the arsenate concentration was increased. In the experiments represented in Table V, the concentrations of both phosphate and arsenate were varied so that several TABLE E$ect of Arsenate on Uptake of Radioactive Phosphate into Adenosine Nucleotide Fraction Arsenate Phosphate WJ c.$%m IV Inorganic P Easily hydrolyzable nucleotide P c.#.nl. +?r px cl.u c.p.m. c.p.m. per pai Flask contents, as final concentrations, 0.01 M L-glutamate, M MgCI,, M NaF, M tris(hydroxymethyl)aminomethane buffer, M adenosinetriphosphate, M (5 PM) phosphate, 1.5 ml. of the enzyme preparation, and arsenate to give the molar ratios shown above. 0.9 per cent KC1 added to make a total of 3 ml. Arsenate was added after 3 minutes temperature equilibration ml. of P32 solution of negligible phosphate content was added 1 minute later. The reaction was terminated 3.25 minutes after addition of P32. determinations at the same phosphate-arsenate ratio would be obtained at different absolute levels of these ions. The data show quite clearly that the inhibitory effect is dependent upon the arsenate-phosphate ratio. Nearly the same rate of incorporation is obtained at the high levels of arsenate as at the low, provided the phosphate concentration is increased to maintain the same ratio. The first line in Table IV shows a rather puzzling phenomenon. In the absence of arsenate an apparently lower specific activity was obtained in the inorganic fraction compared with nucleotide phosphate. As will be shown in the following paper, this is caused by a dilution of the former with the internal mitochondrial phosphate upon termination of the experiment. It has been found (19) that the mitochondrial phosphate does not equilibrate as rapidly with added inorganic phosphate as does nucleotide phosphorus.

8 242 AEROBIC PHOSPHORYLATION TABLE Significance of Molar Ratio of Arsenate to Phosphate for Inhibition of P32 Uptake V Sample No. Phosphate 1 Arsenate Specific actwity of phosphate Total activity of nucleotide Nucleotide phosphorus formed* 1 la 2 2a 3 3a C.).rn. per p tf c.p.m I Flask contents as for Fig. 2, except that adenosine-5-phosphate replaces ATP and the arsenate and phosphate concentrations are as shown above. Arsenate was added after 3 minutes temperature equilibration. P32-labeled phosphate was added 1 minute later. The reaction was terminated 2 minutes after phosphate addition. * Total activity of nucleotide fraction Specific activity of phosphate * Comments The complicating reduction of arsenate to arsenite has made it rather difficult to obtain a clear cut picture. Nevertheless, the data indicate that arsenate may substitute for phosphate in aerobic phosphorylation. The affinity or the accessibility of the phosphorylation system for phosphate, however, appears to be considerably greater than for arsenate. Pronounced effects are seen only if there is a considerable excess of arsenate. Under these conditions, the rate of incorporation of radioactive phosphate into ATP is strongly depressed. This depression of phosphorylation, in conjunction with the stimulation of respiration by arsenate, indicates a disruption of phosphate fixation in the presence of arsenate. This is presumably due to an arsenolysis somewhere in the chain of reactions. PM SUMMARY Arsenate increases the respiration of washed mitochondria in the absence of inorganic phosphate and partially replaces adenine nucleotide. The P: 0 ratio is depressed in the presence of arsenate and, in particular, the incorporation of radioactive phosphate into ATP is markedly diminished. The effect depends on the ratio of arsenate to phosphate and becomes appreciable at ratios above 3 to 4. It is concluded that by substitution for phosphate arsenate disrupts aerobic phosphorylation. The experiments are complicated by a respiratory inhibition due to partial reduction of arsenate to arsenite..

9 R. K. CRANE AND F. LIPMANN 243 A method is described for a convenient separation of adenine nucleotides, including adenosine-5-phosphate, adenosinediphosphate, and adenosinetriphosphate, from inorganic phosphate and from non-nucleotide phosphoric acid esters, with charcoal adsorption. The charcoal adsorbs the nucleotide fraction, while the inorganic phosphate remains in solution. BIBLIOGRAPHY 1. Harden, A., Alcoholic fermentation, Monographs on biochemistry, London (1932). 2. Needham, D. M., and Pillai, R. K., Biochem. J., 31, 1837 (1937). 3. Warburg, O., and Christian, W., Biochem. Z., 303, 40 (1939). 4. Doudoroff, M., Barker, H. A., and Hassid, W. Z., J. Biol. Chem., 170, 147 (1947). 5. Stadtman, E. R., and Barker, H. A., J. Biol. Chem., 184, 769 (1950). 6. Stadtman, E. R., Novelli, G. D., and Lipmann, F., J. Biol. Chem., 191,365 (1951). 7. Green, D. E., Loomis, W. F., and Auerbach, V. H., J. Biol. Chem., 172,389 (1948). 8. Loomis, W. F., and Lipmann, F., J. Biol. Chem., 179, 503 (1949). 9. LePage, G. A., and Umbreit, W. W., in Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and related methods for the study of tissue metabolism, Minneapolis, 165 (1945). 10. Lehninger, A. L., J. Biol. Chem., 178, 625 (1949). 11. Plaut, G. W. E., Kuby, S. A., and Lardy, H. A., J. Biol. Chem., 184, 243 (1950). 12. Cohn, W. E., and Carter, C. E., J. Am. Chem. Sot., 72, 4273 (1950). 13. Fiske, C. H., Proc. Nut. Acad. SC., 20,25 (1934). 14. Solomon, A. K., and Estes, H. D., Rev. Scient. Instruments, 19, 47 (1948). 15. Fiske, C. H., and Subbarow, Y., J. BioZ. Chem., 66, 375 (1925). 16. Lohmann, K., and Jendrassik, L., Biochem. Z., 178, 419 (1926). 17. Pett, L. B., Biochem. J., 27, 1672 (1933). 18. Hunter, F. E., Jr., in McElroy, W. D., and Glass, B., Phosphorus metabolism, Baltimore, 1, 297 (1951). 19. Crane, R. K., and Lipmann, F., J. BioZ. Chem., 201, 245 (1953).

10 THE EFFECT OF ARSENATE ON AEROBIC PHOSPHORYLATION Robert K. Crane and Fritz Lipmann J. Biol. Chem. 1953, 201: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1